Method for reducing pest damage to corn by treating transgenic corn seeds with clothianidin pesticide

ABSTRACT

A method to protect corn against feeding damage by one or more pests includes the treatment of corn seed having a transgenic event that is targeted against at least one of the pests with clothianidin pesticide (clothianidin) in an amount that is effective against the same or another of the one or more pests. Seeds having such protection are also disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a non-provisional of U.S. Patent ApplicationSerial No. 60/238,405, filed on Oct. 6, 2000, and claims prioritythereto.

BACKGROUND OF THE INVENTION

[0002] (1) Field of the Invention

[0003] The present invention relates generally to the control of peststhat cause damage to corn plants by their feeding activities, and moreparticularly to the control of such corn plant pests by the combinationof a corn seed having a transgenic event and the treatment of such seedwith clothianidin pesticide prior to planting the seed.

[0004] (2) Description of the Related Art

[0005] Insects and related arthropods annually destroy an estimated 15%of agricultural crops in the United States and even more than that indeveloping countries. In addition, competing weeds and parasitic andsaprophytic plants account for even more potential yield losses.

[0006] Some of this damage occurs in the soil when plant pathogens,insects and other such soil borne pests attack the seed after planting.In the production of corn, much of the rest of the damage is caused byrootworms—insect pests that feed upon or otherwise damage the plantroots; and by cutworms, European corn borers, and other pests that feedupon or damage the above ground parts of the plant. General descriptionsof the type and mechanisms of attack of pests on agricultural crops areprovided by, for example, Metcalf, in Destructive and Useful Insects,(1962); and Agrios, in Plant Pathology, 3rd Ed., Academic Press (1988).

[0007] Corn is the most important grain crop in the Midwestern UnitedStates. Among the most serious insect pests of corn in this region isthe larval form of three species of Diabrotica beetles. These includethe Western corn rootworm, Diabrotica vergifera vergifera LeConte, theNorthern corn rootworm, Diabrotica berberi Smith and Diabrotica berberiLawrence, and the Southern corn rootworm, Diabrotica undecimpunctatahowardi Barber. In fact, more insecticide is used for the control ofcorn rootworm than for any other pest of corn, and the total acreagetreated is greater than for any other pest in the United States.

[0008] Corn rootworms (CRW) overwinter in the egg stage in fields wherecorn was grown the previous season. The eggs hatch from late May throughJune. If a corn crop is not followed by another corn crop in thesubsequent year, the larvae will die. Accordingly, the impact of cornrootworm is felt most directly in areas where corn is systematicallyfollowed by corn, as is typical in many areas of the Midwestern UnitedStates.

[0009] After hatching, the larvae pass through three larval stages orinstars, during which they feed on the corn root system. About threeweeks is required for completion of the larval stage. Damage to the cornroot system caused by the feeding of larvae is the major cause ofharvest losses in corn due to corn rootworm. Corn plants that fall overand lodge in the field after weakening or destruction of a major part ofthe root system are the cause of a major portion of this loss, sincethis lodged corn cannot be harvested by conventional mechanizedmachinery and is left in the field.

[0010] Following completion of larval development, the larvae transforminto immobile pupae, and thence into the adult beetles that emerge fromthe soil throughout the summer, with the period of emergence dependingupon the growing location. After emergence, the adult beetles feed forabout two weeks before the females start laying eggs. Initially, theadults feed predominantly in the same field from which they emerged, butlater will migrate to other fields. Peak adult activity normally occursin the U.S. Corn Belt during late July or early August in fields plantedto continuous corn, but activity may peak later in first year or latematuring cornfields. Rootworm beetles begin depositing eggs incornfields approximately two weeks after they emerge. (For moreinformation, see, e.g., Corn Rootworms, Field Crops Pest ManagementCircular #16, Ohio Pest Management & Survey Program, The Ohio StateUniversity, Extension Division, Columbus, Ohio; available online atwww.ag.ohio-state.edu/˜ohioline/icm-fact/fc-16.html, Sep. 13, 2000; andMcGahen et al., Corn Insect Control: Corn Rootworm, PENpages number08801502, Factsheet available from Pennsylvania State University, StateCollege, Pa., 1989).

[0011] In present conventional agricultural practice, in cases wherecorn follows corn, it is normal for an insecticide to be applied toprotect the corn root system from severe feeding by rootworm larvae.Conventional practice is to treat for the adult beetles or to treat forthe larvae. Examples of conventional treatment formulations for adultbeetles include the application of carbaryl insecticides (e.g., SEVIN®80S at 1.0-2.0 lbs active/acre); fenvalerate or esfenvalerate (e.g.,PYDRIN® 2.4EC at 0.1 to 0.2 lbs active/acre, or ASANA® 0.66EC at 0.03 to0.05 lbs active/acre); malathion (57% E at 0.9 lbs active/acre);permethrin (e.g., AMBUSH® 2.0EC at 0.1 to 0.2 lbs active/acre, orPOUNCE® 3.2EC at 0.1 to 0.2 lbs active ingredient/acre); or PENNCAP-M®at 0.25-0.5 lbs active/acre.

[0012] To treat for CRW larvae, conventional practice is to apply a soilinsecticide either at or after planting, but preferably as close to egghatching as possible. Conventional treatments include carbofuraninsecticides (e.g., FURADAN® 15G at 8 oz/1000 ft of row); chloropyrifos(e.g., LORSBAN® 15G at 8 oz/1000 ft of row); fonophos (e.g., DYFONATE®20G at 4.5 to 6.0 oz/1000 ft of row); phorate (e.g., THIMET® 20G at 6oz/1000 ft of row); terbufos (e.g., COUNTER® 15G at 8 oz/1000 ft ofrow), or tefluthrin (e.g., FORCE® 3G at 4 to 5 oz/1000 ft of row).

[0013] Many of the chemical pesticides listed above are known to beharmful to humans and to animals in general. The environmental harm thatthese pesticides cause is often exacerbated due to the practice ofapplying the pesticides by foliar spraying or direct application to thesurface of the soil. Wind-drift, leaching, and runoff can cause themigration of a large fraction of the pesticide out of the desired zoneof activity and into surface waters and direct contact with birds,animals and humans.

[0014] Because of concern about the impact of chemical pesticides onpublic health and the health of the environment, significant effortshave been made to find ways to reduce the amount of chemical pesticidesthat are used. Recently, much of this effort has focused on thedevelopment of transgenic crops that are engineered to express insecttoxicants derived from microorganisms. For example, U.S. Pat. No.5,877,012 to Estruch et al. discloses the cloning and expression ofproteins from such organisms as Bacillus, Pseudomonas, Clavibacter andRhizobium into plants to obtain transgenic plants with resistance tosuch pests as black cutworms, armyworms, several borers and other insectpests. Publication WO/EP97/07089 by Privalle et al. teaches thetransformation of monocotyledons, such as corn, with a recombinant DNAsequence encoding peroxidase for the protection of the plant fromfeeding by corn borers, earworms and cutworms. Jansens et al., in CropSci., 37(5):1616-1624 (1997), reported the production of transgenic corncontaining a gene encoding a crystalline protein from Bacillusthuringiensis (Bt) that controlled both generations of the European cornborer. U. S. Pat. Nos. 5,625,136 and 5,859,336 to Koziel et al. reportedthat the transformation of corn with a gene from B. thuringiensis thatencoded for delta-endotoxins provided the transgenic corn with improvedresistance to European corn borer. A comprehensive report of fieldtrials of transgenic corn that expresses an insecticidal protein from B.thuringiensis has been provided by Armstrong et al., in Crop Science,35(2):550-557 (1995).

[0015] It was known that wild-type Bt δ-endotoxins had low activityagainst coleopteran insects, and Kreig et al., in 1983, reported thefirst isolation of a coleopteran-toxic B. thuringiensis strain. (SeeU.S. Pat. No. 4,766,203). U.S. Pat. Nos. 4,797,279 and 4,910,016, alsodisclosed wild-type and hybrid B. thuringiensis strains that producedproteins having some coleopteran activity. More recently, however, moreprecise genetic engineering methods have shown promise in developingmodified B. thuringiensis proteins that have significantly higher levelsof corn rootworm activity than those produced by wild-type parents.(See, e.g., WO 99/31248 to Ecogen, Inc. and Monsanto Company).

[0016] However, it is not known at present whether any transgenic eventalone will be sufficiently effective to protect corn from damage by cornrootworm in heavily infested fields that are dedicated to serial corn.In fact, the total control of corn rootworm damage by any one transgenicevent may not be desirable in the long term, because of the potentialfor the development of resistant strains of the target pest.

[0017] Another alternative to the conventional forms of pesticideapplication is the treatment of plant seeds with pesticides. The use offungicides to protect seeds from attack after planting, and the use oflow levels of insecticides for the protection of, for example, corn seedfrom wireworm, has been used for some time. Seed treatment withpesticides has the advantages providing for the protection of the seeds,while minimizing the amount of pesticide that was required and limitingthe amount of contact with the pesticide and the number of differentfield applications that were necessary.

[0018] Other examples of the control of pests by applying insecticidesdirectly to plant seed are provided in, for example, U.S. Pat. No.5,696,144, which discloses that the European corn borer caused lessfeeding damage to corn plants grown from seed treated with a1-arylpyrazole compound at a rate of 500 g per quintal of seed thancontrol plants grown from untreated seed. In addition, U.S. Pat. No.5,876,739 to Turnblad et al. (and its parent, U.S. Pat. No. 5,849,320)disclose a method for controlling soil-borne insects which involvestreating seeds with a coating containing one or more polymeric bindersand an insecticide. This reference provides a list of insecticides thatit identifies as candidates for use in this coating and also names anumber of potential target insects. However, while the U.S. Pat. No.5,876,739 states that treating corn seed with a coating containing aparticular insecticide protects corn roots from damage by the cornrootworm, it does not indicate or otherwise suggest that such treatmentcould be used with seed having a transgenic event.

[0019] The treatment of seed having a transgenic event withnitroimino-or nitroguanidino-compound pesticides has been mentioned(See, e.g., WO 99/35913), however, no guidance has been found as to thepotential utility or efficacy of such treatments, or the details of howsuch treatments might be effected—such as the amounts of activeingredient that would be necessary per unit amount of seed—and noexamples that would give reason to believe that the proposed treatmentswould actually provide suitable protection.

[0020] Therefore, although recent developments in genetic engineering ofplants have improved the ability to protect plants from pests withoutusing chemical pesticides, and while such techniques as the treatment ofseeds with pesticides have reducing the harmful effects of pesticides onthe environment, numerous problems remain that limit the successfulapplication of these methods under actual field conditions. Accordingly,it would be useful to provide an improved method for the protection ofplants, especially corn plants, from feeding damage by pests. It wouldbe particularly useful if such method would reduce the requiredapplication rate of conventional chemical pesticides, and also if itwould limit the number of separate field operations that were requiredfor crop planting and cultivation.

BRIEF SUMMARY OF THE INVENTION

[0021] Briefly, therefore, the present invention is directed to a novelmethod for protecting a transgenic corn plant against feeding damage byone or more pests, the method comprising providing a seed for thetransgenic corn plant which seed comprises a transgenic event havingactivity against at least one of the one or more pests; and treating theseed with an effective amount of clothianidin pesticide.

[0022] The present invention is also directed to a novel transgenic cornseed that has been treated by providing a seed for the transgenic cornplant which seed comprises a transgenic event having activity against atleast one of the one or more pests, and treating the seed with aneffective amount of clothianidin pesticide.

[0023] The present invention is also directed to a novel seed of atransgenic corn plant that provides increased resistance to theresulting corn plant against feeding damage by one or more pests,comprising a transgenic event having activity against at least one ofthe one or more pests, which seed has been treated with an effectiveamount of clothianidin pesticide.

[0024] Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of a method for theprotection of plants, especially corn plants, from feeding damage bypests; the provision of a method that would reduce the requiredapplication rate of conventional chemical pesticides; and the provisionof a method that would limit the number of separate field operationsthat were required for crop planting and cultivation.

DETAILED DESCRIPTION OF THE INVENTION

[0025] In accordance with the present invention, it has been discoveredthat corn plants can be protected against feeding damage by one or morepests by a method that includes providing a corn seed having atransgenic event that has activity against at least one of the pests andthen treating the transgenic corn seed with an effective amount ofclothianidin pesticide. In preferred embodiments of this invention, ithas been found that the combination of a transgenic event havingactivity against corn rootworm and treatment of the seed withclothianidin provides unexpectedly synergistic advantages to seedshaving such treatment, including unexpectedly superior efficacy forprotection against damage to the resulting corn plant by corn rootworm.In particular, it was found that the combination of the presentinvention was unexpectedly superior to either the transgenic eventalone, or to seed treatment with clothianidin alone, in protecting cornplants against more severe levels of damage by corn rootworm—levels ofdamage that are known to reduce corn yield.

[0026] Corn plants and seeds that have been engineered to includeexogenous genes derived from Bacillus thuringiensis that encode for theexpression of Cry3 67 -endotoxins having activity against Coleopteranpests are known, as are methods for the treatment of seeds (even sometransgenic seeds) with pesticides. However, it had not been realizeduntil the present invention that certain effective amounts ofclothianidin pesticide could be used to treat corn seeds having suchCry3 events, with the result that the combination would be effective,and preferably unexpectedly superior, in increasing the efficacy of boththe pesticide and the transgenic event, and would provide the additionaladvantages of increasing the ability to match pesticidal activityagainst pest pressure, decreasing cost of treatment and/or application,increasing safety of seed handling, and decreasing environmental impactof either or both the event and the pesticide.

[0027] In particular, it has been found in preferred embodiments thatthe treatment of a transgenic corn seeds that are capable of expressingcertain modified Cry3Bb proteins with from about 100 gm to about 400 gmof clothianidin per 100 kg of seed provided unexpectedly superiorprotection against corn rootworm. In addition, it is believed that suchcombination is also effective to protect the emergent corn plantsagainst damage by black cutworm. The seeds of the present invention arealso believed to have the property of decreasing the cost of pesticideuse, because less of the pesticide can be used to obtain a requiredamount of protection than if the innovative method is not used.Moreover, because less pesticide is used and because it is applied priorto planting and without a separate field application, it is believedthat the subject method is therefore safer to the operator and to theenvironment, and is potentially less expensive than conventionalmethods.

[0028] When it is said that some effects are “synergistic”, it is meantto include the synergistic effects of the combination on the pesticidalactivity (or efficacy) of the combination of the transgenic event andthe pesticide. However, it is not intended that such synergistic effectsbe limited to the pesticidal activity, but that they should also includesuch unexpected advantages as increased scope of activity, advantageousactivity profile as related to type and amount of damage reduction,decreased cost of pesticide and application, decreased pesticidedistribution in the environment, decreased pesticide exposure ofpersonnel who produce, handle and plant corn seeds, and other advantagesknown to those skilled in the art.

[0029] The present invention also provides an advantage of increasingthe ability to match pesticidal activity against pest pressure. Thisrefers to the ability to design the combination of the transgenic eventand the pesticide treatment so that the seed or the resulting plant isprovided with effective pesticidal activity during the period whenfeeding pressure from the target pest on the seed or plant reaches itsmaximum. By way of example, when clothianidin is applied to a corn seedhaving a corn rootworm transgenic event, the pesticide can be applied ina coating designed to provide controlled release of the clothianidin.The release rate can be selected so that the clothianidin providesprotection against such other pests as, for example, black cutworm, atthe post emergence stage of corn, while the transgenic event providescorn rootworm protection at a later stage of plant development—when suchprotection is needed.

[0030] As used herein, the terms “pesticidal effect” and “pesticidalactivity”, or “activity” refer to a toxic effect against a pest. Theterms “activity against (one or more) pests”, also have the samemeaning. When it is said that a seed or plant is “protected againstfeeding damage by one or more pests”, it is meant that such seed orplant possesses a feature having direct or indirect action on one ormore pests that results in reduced feeding damage by such pest or pestson the seeds, roots, shoots and foliage of plants having such feature ascompared to the feeding damage caused under the same conditions toplants not having such feature. Such direct or indirect actions includeinducing death of the pest, repelling the pest from the plant seeds,roots, shoots and/or foliage, inhibiting feeding of the pest on, or thelaying of its eggs on, the plant seeds, roots, shoots and/or foliage,and inhibiting or preventing reproduction of the pest.

[0031] The term “insecticidal activity” has the same meaning aspesticidal activity, except it is limited to those instances where thepest is an insect. Except where specifically noted, when the term“pesticide” is used herein, that term refers to a chemical pesticidethat is supplied externally to the seed, and it is not meant to includeactive agents that are produced by the particular seed or the plant thatgrows from the particular seed. However, the terms “pesticidal activity”and “insecticidal activity” can be used with reference to the activityof either, or both, an externally supplied pesticide and/or an agentthat is produced by the seed or the plant.

[0032] One feature of the present invention is a seed of a transgeniccorn plant. As used herein, the terms “transgenic corn plant” mean acorn plant or progeny thereof derived from a transformed corn plant cellor protoplast, wherein the plant DNA contains an introduced exogenousDNA molecule not originally present in a native, non-transgenic plant ofthe same strain.

[0033] The transgenic corn seed is one that contains an exogenous genethat encodes a pesticidal protein. Pesticidal proteins of this type aredescribed by Schnepf et al., in Microbiology & Molecular BiologyReviews, 62:775-806 (1998), and by ffrench-Constant and Bowen, in CMSLCell. Mol. Life Sci., 57:828-833 (2000). In one application of theinvention, the pesticidal protein is an insecticidal protein.

[0034] It is preferred that the seed contains an exogenous gene derivedfrom a strain of Bacillus thuringiensis, and in particular, it ispreferred that the exogenous gene is one that encodes an insecticidalδ-endotoxin derived from B. thuringiensis. Such δ-endotoxins aredescribed in WO 99/31248 and U.S. Pat. No. 6,063,597, and include theCry3 toxins. Nucleic acid segments that encode modified B. thuringiensiscoleopteran-toxic crystal proteins that are useful in the presentinvention are described in U.S. Pat. No. 6,060,594, and insect resistanttransgenic plants that include nucleic acid sequences that encode suchinsecticidal proteins are discussed in U.S. Pat. No. 6,023,013. It ispreferred that the δ-endotoxins of the present invention include theCry3B proteins, and even more preferred that the δ-endotoxins includethe coleopteran-active Cry3Bb proteins. The nomenclature of the B.thuringiensis insecticidal crystal proteins was set forth by Höfte andWhitely, Microbiol. Rev., 53:242-255, 1989. This nomenclature has beenrevised, and the revised nomenclature can be found athttp://epunix.biols.susx.ac.uk/Home/Neil-Crickmore/Bt/index.html. Therevised nomenclature will be used herein to describe transgenic eventfeatures and the δ-endotoxin proteins encoded by the transgenic event.

[0035] When the terms “transgenic event” are used herein, such terms aremeant to refer to the genetically engineered DNA that is describedabove, but also to include the protein(s) that are encoded by themodified gene. A transgenic event in a corn seed, or corn plant,therefore, includes the ability to express a protein. When it is saidthat a “transgenic event has activity against a pest”, it is to beunderstood that it is the protein that is encoded by the gene thatactually has such activity when the protein is expressed and broughtinto contact with the pest.

[0036] WO 99/31248 and U.S. Pat. No. 6,063,597 describe methods forgenetically engineering B. thuringiensis δ-endotoxin genes so thatmodified δ-endotoxins can be expressed. The modified δ-endotoxin differfrom the wild-type proteins by having specific amino acid substitutions,additions or deletions as compared with the proteins produced by thewild-type organism. Such modified δ-endotoxins are identified herein bythe use of an asterisk (*), or by reference to a specific protein by itsidentifying number. Thus, a genetically modified Cry3 δ-endotoxin wouldbe expressed as Cry3*, examples of which include, without limitation:Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234,Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239,Cry3Bb.11241, Cry3Bb.11242, and Cry3Bb.11098.

[0037] Some of the modified δ-endotoxins that were described in WO99/31248 and in U.S. Pat. No. 6,063,597 were found to have enhancedactivity against coleopteran insects, and in particular againstDiabrotica spp., including corn rootworm. As used herein, the terms“enhanced activity” refer to the increased insecticidal activity of amodified toxin as compared with the activity of the same toxin withoutthe amino acid modifications when both are tested under the sameconditions. In particular, it was found that Cry3* δ-endotoxins hadenhanced activity against corn rootworm, and are therefore preferred foruse in the present invention. More preferred are Cry3B* δ-endotoxins,and even more preferred are Cry3Bb* δ-endotoxins. Even more preferredtransgenic events are those that comprise the ability to express themodified δ-endotoxins that are listed in the following table. Also shownin the table are strains of transgenic B. thuringiensis that includegenes for expression of the respective novel endotoxins, and the dateand accession number of their deposit with the Agricultural ResearchService Collection (NRRL) at 1815 N. University Street, Peoria, Ill.91904. ACCESSION NUMBER (NRRL STRAIN DEPOSIT DATE PROTEIN NUMBER)EG11230 5/27/97 Cry3Bb.11230 B-21768 EG11231 5/27/97 Cry3Bb.11231B-21769 EG11232 5/27/97 Cry3Bb.11232 B-21770 EG11233 5/27/97Cry3Bb.11233 B-21771 EG11234 5/27/97 Cry3Bb.11234 B-21772 EG112355/27/97 Cry3Bb.11235 B-21773 EG11236 5/27/97 Cry3Bb.11236 B-21774EG11237 5/27/97 Cry3Bb.11237 B-21775 EG11238 5/27/97 Cry3Bb.11238B-21776 EG11239 5/27/97 Cry3Bb.11239 B-21777 EG11241 5/27/97Cry3Bb.11241 B-21778 EG11242 5/27/97 Cry3Bb.11242 B-21779 EG1109811/28/97 Cry3Bb.11098 B-21903

[0038] It has also been found that a preferred use of the presentinvention is for reducing pest feeding damage when used in combinationwith seeds having transgenic events that have certain levels ofeffectiveness against such pest. To illustrate which levels ofeffectiveness are preferred, the following example will use the IowaRoot Rating Method (Hills and Peters, J. Econ. Entomol., 64:764-765,1971), which measures corn rootworm feeding damage to corn roots on a1-6 scale. In the rating, 1=no damage or only a few minor feeding scars;2=feeding scars evident but no roots eaten off to within 1½ inch of theplant; 3=several roots eaten off to within ½ inch of the plant, butnever the equivalent of an entire node of roots is destroyed; 4=one rootnode completely destroyed; 5=two root nodes completely destroyed; and6=three or more root nodes destroyed. A destroyed root is defined as aroot that has been pruned to within 1½ inch of the base. Pruned roots donot have to originate from a single node, but all pruned roots mustequal the equivalent of a full node to count as a destroyed node.

[0039] As used herein, a transgenic event is within the preferred rangeof effectiveness level against a target pest if that event reducesfeeding damage by that pest by a certain amount as compared with thesame crop without the transgenic event, but does not preventsubstantially all damage by the target pest. For example, if 10% oftransgenic corn suffered corn rootworm damage of 4 or higher on the Iowa1-6 Scale, while 80% of non-transgenic corn suffered damage of 4 orhigher, then it could be said that the damage to the transgenic corn was(10/80)×100=12.5% of that of the non-transgenic corn. For the purposesof the present invention, it will be understood that a transgenic eventin corn is within the preferred range of effectiveness level if cornhaving such event suffers from about 5% to about 50% of the damagesuffered by non-transgenic corn due to the same pest under the sameconditions. It is more preferred that corn having such transgenic eventsuffers from about 10% to about 40% of the damage suffered bynon-transgenic corn by the same pest under the same conditions, evenmore preferred is damage of from about 15% to about 30%, and yet morepreferred is damage of from about 20% to about 30% of the damagesuffered by non-transgenic corn by the same pest under the sameconditions. As used herein, when the term “about” is used to describethe degree of damage to corn, it is to be understood that the degree ofdamage can be above or below the limits described by as much as 1% or 2%and still be considered to be within the ranges described. By way ofexample, a level of 4.5% damage would be regarded as being “about 5%”.

[0040] Without wishing to be bound to this or any other theory, it isbelieved that the pesticidal seed treatment can provide significantadvantages when combined with a transgenic event that providesprotection that is within the preferred effectiveness range against atarget pest. In addition, it is believed that there are situations thatare well known to those having skill in the art, where it isadvantageous to have such transgenic events within the preferred rangeof effectiveness.

[0041] The present invention also includes seeds and plants having morethat one transgenic event. Such combinations are referred to as“stacked” transgenic events. These stacked transgenic events can beevents that are directed at the same target pest, or they can bedirected at different target pests. In one preferred method, a seedhaving the ability to express a Cry 3 protein also has the ability toexpress at least one other insecticidal protein that is different from aCry 3 protein.

[0042] In another preferred method, the seed having the ability toexpress a Cry 3 protein also has a transgenic event that providesherbicide tolerance. It is more preferred that the transgenic event thatprovides herbicide tolerance is an event that provides resistance toglyphosate, N-(phosphonomethyl) glycine, including the isopropylaminesalt form of such herbicide, even more preferred is the transgenic eventthat is effective to provide the herbicide resistance of ROUNDUP READY®plants and seeds available from Monsanto Co., St. Louis, Mo.

[0043] In the present method, a corn seed having a transgenic event istreated with a pesticide that is identified as clothianidin,(N-[(2-chloro-5-thiazoyl)methyl]-N′-methyl-N″-nitro,[C(E)]-(9Cl)-guanidine,CAS RN 210880-92-5, having a developmental number of Tl-435).

[0044] When the insecticide clothianidin is described herein, it is tobe understood that the description is intended to include salt forms ofthe insecticide as well as any isomeric and/or tautomeric form of theinsecticide that exhibits the same insecticidal activity as the form ofthe insecticide that is described. The clothianidin insecticide that isuseful in the present method can be of any grade or purity that pass inthe trade as such insecticide. Other materials that accompany theinsecticide in commercial preparations as impurities can be tolerated inthe subject methods and compositions, as long as such other materials donot destabilize the composition or significantly reduce or destroy theactivity of any of the insecticide components or the transgenic eventagainst the target pest(s). One of ordinary skill in the art of theproduction of insecticides can readily identify those impurities thatcan be tolerated and those that cannot.

[0045] It has been found that the present method is useful to protectseeds and plants against a wide array of agricultural pests, includinginsects, mites, fungi, yeasts, molds and bacteria.

[0046] When an insect is the target pest for the present invention, suchpests include but are not limited to:

[0047] from the order Lepidoptera, for example,

[0048] Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp.,Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp,Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella,Carposina nipponensis, Chilo spp., Choristoneura spp., Clysiaambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp.,Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydiaspp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp.,Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp.,Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis,Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella,Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp.,Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp.,Ostrinia Nubilalis, Pammene spp., Pandemis spp., Panolis flammea,Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pierisspp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp.,Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp.,Tortrix spp., Trichoplusia ni and Yponomeuta spp.;

[0049] from the order Coleoptera, for example,

[0050] Agrotes spp., Anthonomus spp., Atomaria linearis, Chaetocnematibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabroticaspp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata,Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchusspp., Phlyctinus spp., Popillia spp., Psylliodes spp., Rhizopertha spp.,Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebrio spp., Triboliumspp. and Trogoderma spp.;

[0051] from the order Orthoptera, for example,

[0052] Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaeamaderae, Locusta spp., Periplaneta ssp., and Schistocerca spp.;

[0053] from the order Isoptera, for example,

[0054] Reticulitemes ssp;

[0055] from the order Psocoptera, for example,

[0056] Liposcelis spp.;

[0057] from the order Anoplura, for example,

[0058] Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigusspp. and Phylloxera spp.;

[0059] from the order Mallophaga, for example,

[0060] Damalinea spp. and Trichodectes spp.;

[0061] from the order Thysanoptera, for example,

[0062] Franklinella spp., Hercinothrips spp., Taeniothrips spp., Thripspalmi, Thrips tabaci and Scirtothrips aurantii;

[0063] from the order Heteroptera, for example,

[0064] Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistusspp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp.,Rhodnius spp., Sahlbergella singularis, Scotinophara spp. and Triatomaspp.;

[0065] from the order Homoptera, for example,

[0066]Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp.,Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplasterspp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccushesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp.,Gascardia spp., Laodelphax spp., Lacanium corni, Lepidosaphes spp.,Macrosiphus spp., Myzus spp., Nehotettix spp., Nilaparvata spp.,Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp.,Pseudococcus spp., Psylla ssp., Pulvinaria aethiopica, Quadraspidiotusspp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphisspp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae andUnaspis citri;

[0067] from the order Hymenoptera, for example,

[0068] Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae,Gilpinia polytoma, Hoplocampa spp., Lasius sppp., Monomorium pharaonis,Neodiprion spp, Solenopsis spp. and Vespa ssp.;

[0069] from the order Diptera, for example,

[0070] Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphoraerythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebraspp., Dacus spp., Drosophila melanogaster, Fannia spp., Gastrophilusspp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomysa spp.,Lucilia spp., Melanagromyza spp., Musca ssp., Oestrus spp., Orseoliaspp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletispomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. andTipula spp.,

[0071] from the order Siphonaptera, for example,

[0072] Ceratophyllus spp. und Xenopsylla cheopis and

[0073] from the order Thysanura, for example,

[0074]Lepisma saccharina.

[0075] It has been found that the present invention is particularlyeffective when the insect pest is a Diabrotica spp., and especially whenthe pest is Diabrotica virgifera, Diabrotica barberi, or Diabroticaundecimpunctata.

[0076] In the method of the present invention, clothianidin is appliedto a transgenic corn seed. Although it is believed that the presentmethod can be applied to a transgenic corn seed in any physiologicalstate, it is preferred that the seed be in a sufficiently durable statethat it incurs no damage during the treatment process. Typically, theseed would be a seed that had been harvested from the field; removedfrom the plant; and separated from any cob, stalk, outer husk, andsurrounding pulp or other non-seed plant material. The seed wouldpreferably also be biologically stable to the extent that the treatmentwould cause no biological damage to the seed. In one embodiment, forexample, the treatment can be applied to seed corn that has beenharvested, cleaned and dried to a moisture content below about 15% byweight. In an alternative embodiment, the seed can be one that has beendried and then primed with water and/or another material and thenre-dried before or during the treatment with the pesticide. Within thelimitations just described, it is believed that the treatment can beapplied to the seed at any time between harvest of the seed and sowingof the seed. As used herein, the term “unsown seed” is meant to includeseed at any period between the harvest of the seed and the sowing of theseed in the ground for the purpose of germination and growth of theplant.

[0077] When it is said that unsown seed is “treated” with the pesticide,such treatment is not meant to include those practices in which thepesticide is applied to the soil, rather than to the seed. For example,such treatments as the application of the pesticide in bands, “T”-bands,or in-furrow, at the same time as the seed is sowed are not consideredto be included in the present invention.

[0078] The pesticide, or combination of pesticides, can be applied“neat”, that is, without any diluting or additional components present.However, the pesticide is typically applied to the seeds in the form ofa pesticide formulation. This formulation may contain one or more otherdesirable components including but not limited to liquid diluents,binders to serve as a matrix for the pesticide, fillers for protectingthe seeds during stress conditions, and plasticizers to improveflexibility, adhesion and/or spreadability of the coating. In addition,for oily pesticide formulations containing little or no filler, it maybe desirable to add to the formulation drying agents such as calciumcarbonate, kaolin or bentonite clay, perlite, diatomaceous earth or anyother adsorbent material. Use of such components in seed treatments isknown in the art. See, e.g., U.S. Pat. No. 5,876,739. The skilledartisan can readily select desirable components to use in the pesticideformulation depending on the seed type to be treated and the particularpesticide that is selected. In addition, readily available commercialformulations of known pesticides may be used, as demonstrated in theexamples below.

[0079] The seeds may also be treated with one or more of the followingingredients: other pesticides, including compounds which act only belowthe ground; fungicides, such as captan, thiram, metalaxyl,(methoxam=resolved isomer of metalaxyl), fludioxonil, oxadixyl, andisomers of each of those materials, and the like; herbicides, includingcompounds selected from carbamates, thiocarbamates, acetamides,triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils,phenoxys, ureas, and benzoic acids; herbicidal safeners such asbenzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide,various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone,naphthalic anhydride compounds, and oxime derivatives; fertilizers; andbiocontrol agents such as naturally-occurring or recombinant bacteriaand fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia,Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. Theseingredients may be added as a separate layer on the seed oralternatively may be added as part of the pesticide composition.

[0080] Preferably, the amount of the novel composition or otheringredients used in the seed treatment should not inhibit generation ofthe seed, or cause phytotoxic damage to the seed.

[0081] The pesticide formulation that is used to treat the transgeniccorn seed in the present invention can be in the form of a suspension;emulsion; slurry of particles in an aqueous medium (e.g., water);wettable powder; wettable granules (dry flowable); and dry granules. Ifformulated as a suspension or slurry, the concentration of the activeingredient in the formulation is preferably about 0.5% to about 99% byweight (w/w), preferably 5-40%.

[0082] As mentioned above, other conventional inactive or inertingredients can be incorporated into the formulation. Such inertingredients include but are not limited to: conventional stickingagents, dispersing agents such as methylcellulose (Methocel A15LV orMethocel A15C, for example, serve as combined dispersant/sticking agentsfor use in seed treatments), polyvinyl alcohol (e.g., Elvanol 51-05),lecithin (e.g., Yelkinol P), polymeric dispersants (e.g.,polyvinylpyrrolidone/vinyl acetate PVP/VA S-630), thickeners (e.g., claythickeners such as Van Gel B to improve viscosity and reduce settling ofparticle suspensions), emulsion stabilizers, surfactants, antifreezecompounds (e.g., urea), dyes, colorants, and the like. Further inertingredients useful in the present invention can be found inMcCutcheon's, vol. 1, “Emulsifiers and Detergents,” MC PublishingCompany, Glen Rock, N.J., U.S.A., 1996. Additional inert ingredientsuseful in the present invention can be found in McCutcheon's, vol. 2,“Functional Materials,” MC Publishing Company, Glen Rock, N.J., U.S.A.,1996.

[0083] The pesticides and pesticide formulations of the presentinvention can be applied to seeds by any standard seed treatmentmethodology, including but not limited to mixing in a container (e.g., abottle or bag), mechanical application, tumbling, spraying, andimmersion. Any conventional active or inert material can be used forcontacting seeds with pesticides according to the present invention,such as conventional film-coating materials including but not limited towater-based film coating materials such as Sepiret (Seppic, Inc.,Fairfield, N.J.) and Opacoat (Berwind Pharm. Services, Westpoint, Pa.).

[0084] The subject pesticides can be applied to a seed as a component ofa seed coating. Seed coating methods and compositions that are known inthe art are useful when they are modified by the addition of one of theembodiments of the combination of pesticides of the present invention.Such coating methods and apparatus for their application are disclosedin, for example, U.S. Pat. Nos. 5,918,413, 5,891,246, 5,554,445,5,389,399, 5,107,787, 5,080,925, 4,759,945 and 4,465,017. Seed coatingcompositions are disclosed, for example, in U.S. Pat. Nos. 5,939,356,5,882,713, 5,876,739, 5,849,320, 5,834,447, 5,791,084, 5,661,103,5,622,003, 5,580,544, 5,328,942, 5,300,127, 4,735,015, 4,634,587,4,383,391, 4,372,080, 4,339,456, 4,272,417 and 4,245,432, among others.

[0085] Useful seed coatings contain one or more binders and at least oneof the subject combinations of pesticides.

[0086] Binders that are useful in the present invention preferablycomprise an adhesive polymer that may be natural or synthetic and iswithout phytotoxic effect on the seed to be coated. The binder may beselected from polyvinyl acetates; polyvinyl acetate copolymers; ethylenevinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcoholcopolymers; celluloses, including ethylcelluloses, methylcelluloses,hydroxymethylcelluloses, hydroxypropylcelluloses andcarboxymethylcellulose; polyvinylpyrolidones; polysaccharides, includingstarch, modified starch, dextrins, maltodextrins, alginate andchitosans; fats; oils; proteins, including gelatin and zeins; gumarabics; shellacs; vinylidene chloride and vinylidene chloridecopolymers; calcium lignosulfonates; acrylic copolymers;polyvinylacrylates; polyethylene oxide; acrylamide polymers andcopolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; andpolychloroprene.

[0087] It is preferred that the binder be selected so that it can serveas a matrix for the subject pesticides. While the binders disclosedabove may all be useful as a matrix, the specific binder will dependupon the properties of the combination of pesticides. The term “matrix”,as used herein, means a continuous solid phase of one or more bindercompounds throughout which is distributed as a discontinuous phase oneor more of the subject pesticides. Optionally, a filler and/or othercomponents can also be present in the matrix. The term matrix is to beunderstood to include what may be viewed as a matrix system, a reservoirsystem or a microencapsulated system. In general, a matrix systemconsists of pesticides of the present invention and filler uniformlydispersed within a polymer, while a reservoir system consists of aseparate phase comprising the subject pesticides, that is physicallydispersed within a surrounding, rate-limiting, polymeric phase.Microencapsulation includes the coating of small particles or dropletsof liquid, but also to dispersions in a solid matrix.

[0088] The amount of binder in the coating can vary, but will be in therange of about 0.01 to about 25% of the weight of the seed, morepreferably from about 0.05 to about 15%, and even more preferably fromabout 0.1% to about 10%.

[0089] As mentioned above, the matrix can optionally include a filler.The filler can be an absorbent or an inert filler, such as are known inthe art, and may include woodflours, clays, activated carbon, sugars,diatomaceous earth, cereal flours, fine-grain inorganic solids, calciumcarbonate, and the like. Clays and inorganic solids, which may be used,include calcium bentonite, kaolin, china clay, talc, perlite, mica,vermiculite, silicas, quartz powder, montmorillonite and mixturesthereof. Sugars, which may be useful, include dextrin and maltodextrin.Cereal flours include wheat flour, oat flour and barley flour.

[0090] The filler is selected so that it will provide a propermicroclimate for the seed, for example the filler is used to increasethe loading rate of the active ingredients and to adjust thecontrol-release of the active ingredients. The filler can aid in theproduction or process of coating the seed. The amount of filler canvary, but generally the weight of the filler components will be in therange of about 0.05 to about 75% of the seed weight, more preferablyabout 0.1 to about 50%, and even more preferably about 0.5% to 15%.

[0091] The pesticides that are useful in the coating are thosepesticides that are described herein. The amount of pesticide that isused for the treatment of the seed will vary depending upon the type ofseed and the type of active ingredients, but the treatment will comprisecontacting the seeds with an amount of the combination of pesticidesthat is pesticidally effective. When insects are the target pest, thatamount will be an amount of the insecticide that is insecticidallyeffective. As used herein, an insecticidally effective amount means thatamount of insecticide that will kill insect pests in the larvae or pupalstate of growth, or will consistently reduce or retard the amount ofdamage produced by insect pests.

[0092] In general, the amount of clothianidin that is applied to theseed in the treatment will range from about 10 gm to about 2000 gm ofthe active ingredient of the pesticide per 100 kg of the weight of theseed. Preferably, the amount of pesticide will be within the range ofabout 50 gm to about 1000 gm active per 100 kg of seed, more preferablywithin the range of about 100 gm to about 600 gm active per 100 kg ofseed, and even more preferably within the range of about 200 gm to about500 gm of active per 100 kg of seed weight. Alternatively, it has beenfound to be preferred that the amount of the pesticide be over about 60gm of the active ingredient of the pesticide per 100 kg of the seed, andmore preferably over about 80 gm per 100 kg of seed.

[0093] The pesticide that is used in the treatment must not inhibitgermination of the seed and should be efficacious in protecting the seedand/or the plant during that time in the target insect's life cycle inwhich it causes injury to the seed or plant. In general, the coatingwill be efficacious for approximately 0 to 120 days after sowing.

[0094] The pesticide of the subject invention can be applied to the seedin the form of a coating. The use of a coating is particularly effectivein accommodating high pesticidal loads, as can be required to treattypically refractory pests, such as corn rootworm, while at the sametime preventing unacceptable phytotoxicity due to the increasedpesticidal load.

[0095] Optionally, a plasticizer can be used in the coating formulation.Plasticizers are typically used to make the film that is formed by thecoating layer more flexible, to improve adhesion and spreadability, andto improve the speed of processing. Improved film flexibility isimportant to minimize chipping, breakage or flaking during storage,handling or sowing processes. Many plasticizers may be used, however,useful plasticizers include polyethylene glycol, glycerol,butylbenzylphthalate, glycol benzoates and related compounds. The rangeof plasticizer in the coating layer will be in the range of from bout0.1 to about 20% by weight.

[0096] When the pesticide used in the coating is an oily typeformulation and little or no filler is present, it may be useful tohasten the drying process by drying the formulation. This optional stepmay be accomplished by means will known in the art and can include theaddition of calcium carbonate, kaolin or bentonite clay, perlite,diatomaceous earth, or any absorbent material that is added preferablyconcurrently with the pesticidal coating layer to absorb the oil orexcess moisture. The amount of calcium carbonate or related compoundsnecessary to effectively provide a dry coating will be in the range ofabout 0.5 to about 10% of the weight of the seed.

[0097] The coatings formed with the pesticide are preferably of the typethat are capable of effecting a slow rate of release of the pesticide bydiffusion or movement through the matrix to the surrounding medium.

[0098] In addition to the coating layer, the seed may be treated withone or more of the following ingredients: other pesticides includingfungicides and herbicides; herbicidal safeners; fertilizers and/orbiocontrol agents. These ingredients may be added as a separate layer oralternatively may be added in the pesticidal coating layer.

[0099] The pesticide formulation may be applied to the seeds usingconventional coating techniques and machines, such as fluidized bedtechniques, the roller mill method, rotostatic seed treaters, and drumcoaters. Other methods, such as spouted beds may also be useful. Theseeds may be presized before coating. After coating, the seeds aretypically dried and then transferred to a sizing machine for sizing.Such procedures are known in the art.

[0100] The pesticide-treated seeds may also be enveloped with a filmovercoating to protect the pesticide coating. Such overcoatings areknown in the art and may be applied using conventional fluidized bed anddrum film coating techniques.

[0101] In another embodiment of the present invention, a pesticide canbe introduced onto or into a seed by use of solid matrix priming. Forexample, a quantity of the pesticide can be mixed with a solid matrixmaterial and then the seed can be placed into contact with the solidmatrix material for a period to allow the pesticide to be introduced tothe seed. The seed can then optionally be separated from the solidmatrix material and stored or used, or the mixture of solid matrixmaterial plus seed can be stored or planted directly. Solid matrixmaterials which are useful in the present invention includepolyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea,polyacrylate, or any other material capable of absorbing or adsorbingthe pesticide for a time and releasing that pesticide into or onto theseed. It is useful to make sure that the pesticide and the solid matrixmaterial are compatible with each other. For example, the solid matrixmaterial should be chosen so that it can release the pesticide at areasonable rate, for example over a period of minutes, hours, or days.

[0102] The present invention further embodies imbibition as anothermethod of treating seed with the pesticide. For example, plant seed canbe combined for a period of time with a solution comprising from about1% by weight to about 75% by weight of the pesticide in a solvent suchas water. Preferably the concentration of the solution is from about 5%by weight to about 50% by weight, more preferably from about 10% byweight to about 25% by weight. During the period that the seed iscombined with the solution, the seed takes up (imbibes) a portion of thepesticide. Optionally, the mixture of plant seed and solution can beagitated, for example by shaking, rolling, tumbling, or other means.After imbibition, the seed can be separated from the solution andoptionally dried, for example by patting or air drying.

[0103] In yet another embodiment, a powdered pesticide can be mixeddirectly with seed. Optionally, a sticking agent can be used to adherethe powder to the seed surface. For example, a quantity of seed can bemixed with a sticking agent and optionally agitated to encourage uniformcoating of the seed with the sticking agent. The seed coated with thesticking agent can then be mixed with the powdered pesticide. Themixture can be agitated, for example by tumbling, to encourage contactof the sticking agent with the powdered pesticide, thereby causing thepowdered pesticide to stick to the seed.

[0104] The present invention also provides a transgenic corn seed thathas been treated with a pesticide by the method described above.

[0105] The treated seeds of the present invention can be used for thepropagation of corn plants in the same manner as conventional treatedcorn seed. The treated seeds can be stored, handled, sowed and tilled inthe same manner as any other pesticide treated seed. Appropriate safetymeasures should be taken to limit contact of the treated seed withhumans, food or feed materials, water and birds and wild or domesticanimals.

[0106] Preferred embodiments of the invention are described in thefollowing examples. Other embodiments within the scope of the claimsherein will be apparent to one skilled in the art from consideration ofthe specification or practice of the invention as disclosed herein. Itis intended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

[0107] The following examples describe preferred embodiments of theinvention. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples. In the examplesall percentages are given on a weight basis unless otherwise indicated.

EXAMPLE 1

[0108] Production of transgenic corn seed and treatment withclothianidin.

[0109] Corn seeds were prepared to express the Bacillus thuringiensesendotoxin Cry3Bb.11231 by the method described in WO 99/31248 or U.S.Pat. No. 6,023,013.

[0110] Corn seeds of the same hybrid species, with and without thetransgenic event, were treated with clothianidin pesticide as follows. Aseed treatment formulation was prepared by mixing a measured amount ofclothianidin in water as a carrier and applying the formulation for oneminute at room temperature to a measured weight of corn seed in arotostatic seed treater. The respective weights of the pesticidepreparation and the corn seed were calculated to provide the desiredrate of treatment of pesticide on the seed. The pesticide was mixed intosufficient water to permit efficient distribution of the formulation toall of the seeds in the batch while minimizing loss of treatmentformulation due to lack of uptake of the formulation by the seeds.Treated seeds were allowed to sit uncapped for at least four hoursbefore planting.

EXAMPLE 2

[0111] Field trials for the determination of efficacy of transgenicevent Cry3Bb.11231 in corn seed in combination with clothianidin seedtreatments against western and northern corn root worm.

[0112] A field trial was run in accordance with pertinent protocols andin conformance with USDA notification requirements. The purpose of thetrial was to determine the efficacy of transgenic event Cry3Bb.11231 incorn seed in combination with corn rootworm seed treatments usingclothianidin.

[0113] For each growing site that was selected, the plot design includedthe following: Row spacing: 30 inches Plot size: 1 row × 20 feetPlanting density: 1.5-2.0 seed/foot, depending upon location Hybridused: MO17 X A1 Replicates: 4 Design: Randomized complete blockLocations: 5 Larvae source: natural infestations supplemented byartificial infestation of corn rootworm eggs at 1200 eggs/ft (growthstage V2)

[0114] The following seed treatment combinations were used for eachgrowing area: Pesticide and amount No. Corn Seed Type (grams Al/100 kgseed) 1 Isohybrid 2 Cry3Bb.11231 3 Isohybrid Clothianidin @ 100 gmAl/100 kg 4 Isohybrid Clothianidin @ 300 gm Al/100 kg 5 IsohybridClothianidin @ 400 gm Al/100 kg 6 CryBb.11231 Clothianidin @ 100 gmAl/100 kg 7 CryBb.11231 Clothianidin @ 300 gm Al/100 kg 8 IsohybridStandard treatment of Force ® 3G @ 0.15 oz Al/1000 ft row, applied as a5″ band on the soil surface at the time of planting.

[0115] All seed treatments with pesticides were carried out as describedin Example 1. Seed receiving treatment numbers 1 and 2 had no pesticidetreatment that would be expected to be effective against corn rootworm.

[0116] For seeds having treatments numbered 3 though 7, the pesticideswere applied by the methods described in Example 1. In seed treatmentnumber 8, commercially available Force® 3G was applied to the soil in a5″ band at the time of sowing. The levels of application are as shownand are within the ranges recommended for standard commercial practice.

[0117] Corn seeds to be tested were planted and grown at five differentlocations across two Midwestern states in the United States corn beltaccording to the protocol described above.

[0118] The determination of damage by corn rootworm was made accordingto the following protocol. At stage VT-R1, an evaluation of cornrootworm damage was carried out by methods that are well known in theindustry, and damage by corn rootworm was reported according to the Iowa1-6 rating system. In that system, the root systems of 10 corn plantsper plot are recovered and scored using the 1-6 rating scale, where:1=no injury or only a few minor feeding scars, 2=feeding injury evident,but no roots eaten back to 1 ½ inches of the plant, 3=at least one rooteaten off to within 1 ½ inches of the plant, but never an entire node ofroots destroyed, 4=one node of roots eaten back to within 1 ½ inches ofthe plant, 5=two nodes (circles) of roots eaten back to within 1 ½inches of the plant, 6=three nodes (circles) of roots eaten back towithin 1 ½ inches of the plant. TABLE 1 Corn root worm damage toisohybrid corn plants and corn plants having transgenic eventCry3Bb.11231 alone and in combination with seed treatment withclothianidin at seven growing locations during first year trial. CORNROOT WORM DAMAGE IN EACH IOWA CLASS (IOWA 1-6 SCALE) MEANS ACROSSTREATMENT 1 2 3 4 5 6 LOCATIONS Untreated control 0  0  32 74 44 27 4.4Cry3.Bb.11231 5 45 109 16  4  1 2.9 Clothianidin @ 0  6  45 98 23  5 3.9100 g/100 kg Clothianidin @ 0 16  94 60 16  2 3.4 300 g/100 kgClothianidin @ 1 34 113 37  1  0 3.0 400 g/100 kg Cry3Bb.11231 1 55  8816  1  0 2.8 with Clothianidin @ 100 g/100 kg Cry3Bb.11231 2 80  90 16 2  0 2.7 with Clothianidin @ 300 g/100 kg FORCE ® 3G 4 89  66 10  5  52.7 surface band at planting

[0119] The data showed that both the transgenic event and clothianidinprovided some level of protection against corn rootworm damage. Athigher levels of damage (i.e., damage levels 4-6), the transgenic eventsuffered 14.5% of the damage of the non-transgenic control. Therefore,the Cry3Bb.11231 event is considered to be within a preferredeffectiveness range.

[0120] Clothianidin was effective against corn rootworm damage at alllevels tested, but the effectiveness of clothianidin at all levels wasless than the effectiveness of the transgenic event alone. Allcombinations of treatment with clothianidin of the transgenic seed weremore effective against rootworm damage than any pesticide treatmentalone or the transgenic event alone. The combination of Cry3Bb.11231with clothianidin at 300 gm/100 kg of seed provided essentially the sameprotection as the commercial standard treatment of 4.25 gm FORCE® 3G per100 ft of row applied as a surface band at planting. Treatment oftransgenic seed with only 100 gm/100 kg of clothianidin provided almostthe same level of protection.

[0121] The advantages of the present treatment of transgenic seed withclothianidin include the simplification of planting, by removing therequirement for separate application of the pesticide. Furthermore,planting is easier and safer, since the planter does not have to handlea concentrated pesticide.

[0122] The combinations of clothianidin seed treatment with corn seedhaving a Cry3Bb.11231 transgenic event were tested for possible synergyat three levels of rootworm damage. In the first test, shown in Table2(a), the percentage of test plants having damage levels of from 3 to 6,on the Iowa 1-6 Scale, were determined for the control and for seedstreated with the pesticide at two levels, and for seeds having thetransgenic event, alone and in combination. The following formula wasthen used to calculate a “synergy threshold”:

[0123] (% of control Cry3Bb.11231)*(% of control clothianidintreatment)/100.

[0124] This threshold was compared against the percent of control forthe treatment combinations (i.e., Cry3Bb.11231 with clothianidin @ 100gm/100 kg and Cry3Bb.11231 with clothianidin @ 300 gm/100 kg). If thetreatment combination percent of control was below the threshold, thenit was concluded that there was synergy. If the treatment combinationpercent of control was above the threshold, then it was concluded thatsynergy was not demonstrated for that combination. This calculation wasrepeated for damage levels 4-6 and 5-6, and the results of thecalculations are shown in Tables 2(b) and 2(c).

[0125] It was believed that the measurement of rootworm damage at higherdamage levels (i.e., levels 3-6, levels 4-6 and levels 5 and 6) is auseful indicator that correlates with subsequent yield loss due to suchdamage. The reason for this is that rootworm damage at levels 1 and 2seldom causes corn plants to fall over and lodge, and such minimal rootloss is not believed to reduce the number or weight of kernels per ear.However, root damage at levels of 3 and above increasingly causeslodging and loss of yield. Therefore, it is believed that the summeddamage levels of 3-6, 4-6 and 5 and 6, provides a useful indication ofthe effect of corn rootworm damage on subsequent corn yield.

[0126] Table 2(a). Efficacy of seed treatment with two levels ofclothianidin alone and in combination with corn transgenic eventCry3Bb.11231 against corn rootworm damage at levels 3-6 on the Iowa 1-6Scale. PERCENT PLANTS PERCENT HAVING OF THRESHOLD TREATMENT 3-6 DAMAGELEVEL CONTROL SYNERGY Untreated 100 100 — Control Cry3Bb.1123 72.2 72.2— 1 Clothianidin 96.6 96.6 — @ 100 gm/100 kg Clothianidin 91.5 91.5 — @300 gm/100 kg Cry3Bb.1123 65.2 65.2 69.8 1 with clothianidin @ 100gm/100 kg Cry3Bb.1123 56.8 56.8 66.1 1 with clothianidin @ 300 gm/100 kg

[0127] Table 2(b). Efficacy of seed treatment with two levels ofclothianidin alone and in combination with corn transgenic eventCry3Bb.11231 against corn rootworm damage at levels 4-6 on the Iowa 1-6Scale.

[0128] Table 2(c). Efficacy of seed treatment with two levels ofclothianidin alone and in combination with corn transgenic eventCry3Bb.11231 against corn rootworm damage at levels 5-6 on the Iowa 1-6Scale.

[0129] This analysis indicated that the combination of the cornCry3Bb.11231 transgenic event with seed treatment with clothianidin ateither 100 gm/100 kg or 300 gm/100 kg was synergistic and unexpectedlyefficacious against corn rootworm damage at the 3-6 level. At the 4-6level of damage, neither level of clothianidin in combination with thetransgenic event indicated synergy, although the combination of the lowlevel of clothianidin treatment (100 gm/100 kg of seed) showed a levelof protection that approached that needed to show synergy. At the 5-6level of damage, the combination of the lower level of clothianidintreatment (100 gm/100 kg of seed) with the Cry3Bb.11231 event indicatedsynergy, while the combination at the higher level of clothianidintreatment did not. However, damage at the 5-6 level was very low for anyof the combinations that were tested, and was far below that suffered bynon-transgenic corn treated with conventional surface banding (about5.6% damage at the 5-6 levels).

[0130] Accordingly, it was concluded that the combination of thetransgenic event with the clothianidin seed treatment providedsignificant advantages over the use of either method alone, and thatsuch protection was unexpectedly superior in efficacy against severedamage by corn rootworm.

[0131] The discussion of the references herein is intended merely tosummarize the assertions made by their authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

[0132] In view of the above, it will be seen that the several advantagesof the invention are achieved and other advantageous results attained.

[0133] As various changes could be made in the above methods andcompositions without departing from the scope of the invention, it isintended that all matter contained in the above description shall beinterpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. A method for protecting a transgenic corn plantagainst feeding damage by one or more pests, the method comprisingproviding a seed for the transgenic corn plant which seed comprises atransgenic event having activity against at least one of the one or morepests; and treating the seed with an effective amount of clothianidinpesticide.
 2. The method according to claim 1 wherein the transgenicevent comprises the ability to express an insecticidal protein.
 3. Themethod according to claim 1 wherein the transgenic event comprises theability to express a Cry protein.
 4. The method according to claim 3wherein the transgenic event comprises the ability to express a Cry 3protein.
 5. The method according to claim 4 wherein the transgenic eventcomprises the ability to express a Cry3* protein.
 6. The methodaccording to claim 5 wherein the Cry3* protein is a Cry3B* protein. 7.The method according to claim 6 wherein the Cry 3B* protein is selectedfrom the group consisting of Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232,Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237,Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241, Cry3Bb.11242, andCry3Bb.11098.
 8. The method according to claim 7 wherein the Cry 3B*protein is Cry3Bb.11231.
 9. The method according to claim 7 wherein theCry3B* protein is Cry3Bb.11098.
 10. The method according to claim 4wherein the transgenic event is capable of reducing the damage caused bycorn rootworm so that the damage to transgenic corn is within the rangeof from about 5% to about 50% of the damage to non-transgenic corn underthe same conditions, when such damage is expressed as the percent ofcorn plants having a score of 4-6 as measured by the Iowa Corn Rootworm1-6 Scale.
 11. The method according to claim 10 wherein the transgenicevent is capable of reducing the damage caused by corn rootworm so thatthe damage to transgenic corn is within the range of from about 10% toabout 40% of the damage to non-transgenic corn under the sameconditions, when such damage is expressed as the percent of corn plantshaving a score of 4-6 as measured by the Iowa Corn Rootworm 1-6 Scale.12. The method according to claim 11 wherein the transgenic event iscapable of reducing the damage caused by corn rootworm so that thedamage to transgenic corn is within the range of about 15% to about 30%of the damage to non-transgenic corn under the same conditions, whensuch damage is expressed as the percent of corn plants having a score of4-6 as measured by the Iowa Corn Rootworm 1-6 Scale.
 13. The methodaccording to claim 11 wherein the transgenic event is capable ofreducing the damage caused by corn rootworm so that the damage totransgenic corn is within the range of about 20% to about 30% of thedamage to non-transgenic corn under the same conditions, when suchdamage is expressed as the percent of corn plants having a score of 4-6as measured by the Iowa Corn Rootworm 1-6 Scale.
 14. The methodaccording to claim 4 wherein the seed having the ability to express aCry 3 protein also has the ability to express at least one otherinsecticidal protein that is different from a Cry 3 protein.
 15. Themethod according to claim 4 wherein the seed having the ability toexpress a Cry 3 protein also has a transgenic event that providesherbicide tolerance.
 16. The method according to claim 15 wherein thetransgenic event that provides herbicide tolerance against glyphosate.17. The method according to claim 4 wherein the transgenic event hasactivity against an insect.
 18. The method according to claim 17 whereinthe insect is selected from the group consisting of members of theorders of Lepidoptera, Coleoptera and Hemiptera.
 19. The methodaccording to claim 18 wherein the insect is a Coleopteran insect. 20.The method according to claim 19 wherein the insect is a Diabrotica spp.21. The method according to claim 20 wherein the insect comprises atleast one member selected from the group consisting of Diabroticavirgifera, Diabrotica barberi and Diabrotica undecimpunctata.
 22. Themethod of claim 4 wherein the effective amount of clothianidin is fromat least about 10 grams to about 2000 grams of the pesticide activeingredient per 100 kilograms of the seed.
 23. The method of claim 22wherein the effective amount of clothianidin is from at least about 70grams to about 1000 grams of the pesticide active ingredient per 100kilograms of the seed.
 24. The method of claim 23 wherein the effectiveamount of clothianidin is from at least about 100 grams to about 600grams of the pesticide active ingredient per 100 kilograms of the seed.25. The method of claim 24 wherein the effective amount of clothianidinis from at least about 200 grams to about 500 grams of the pesticideactive ingredient per 100 kilograms of the seed.
 26. A transgenic cornseed that has been treated by the method of claim
 1. 27. The transgeniccorn seed according to claim 26 wherein the effective amount ofclothianidin is from at least about 200 grams to about 500 grams of thepesticide active ingredient per 100 kilograms of the seed.
 28. A seed ofa transgenic corn plant that provides increased resistance to theresulting corn plant against feeding damage by one or more pests,comprising a transgenic event having activity against at least one ofthe one or more pests, which seed has been treated with an effectiveamount of clothianidin pesticide.
 29. The seed of claim 28 wherein thetransgenic event is the capability to express an insecticidal protein.30. The seed of claim 29 wherein the transgenic event is the capabilityto express a Cry protein.
 31. The seed of claim 30 wherein thetransgenic event is the capability to express a Cry3 protein derivedfrom Bacillus thuringiensis.
 32. The seed of claim 31 wherein thetransgenic event is the capability to express a Cry3Bb protein derivedfrom Bacillus thuringiensis.
 33. The seed of claim 32 wherein thetransgenic event is the capability to express a Cry3Bb.11230,Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235,Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241,Cry3Bb.11242, or a Cry3Bb.11098 protein derived from Bacillusthuringiensis.